JP2014172899A - Bio activation agent and composition thereof, and method of manufacturing it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve a problem that there are mere agents exerting life science action and/or chemical action.SOLUTION: There is provided a hair growing agent, in which cord blood and/or placental blood is distributed at 50,000 to 400,000 ppm, more preferably at 150,000 to 250,000 ppm, in isotonic solution for micro needle injection, water solution having within ±15% isotonicity, and/or a solution containing one and/or two or more components selected from a group consisting of mannitol, sucrose, gelatin, gelatin lysate, casein, casein lysate, provitamin C, provitamin E as a cell membrane stabilizing agent.

Description

  The present invention relates to a bioactive agent that can be used as a hair growth agent, its composition, and a production method.

  It is known that not only hair roots but also stem cells play an important role in hair growth. Conventionally, various drugs have been researched and developed as hair growth agents.

JP 2000-69961 A

  However, there is a problem that all of them are only drugs that exhibit life science effects and / or chemical effects.

In order to solve the above problems, the present invention has the following configuration.
A first invention is a hair growth agent characterized by containing umbilical cord blood.
2nd invention is the hair growth agent characterized by containing the plasma which removed the blood cell component among cord blood.
A third invention is a hair growth agent characterized by comprising serum from which blood cell components and fiber proteins such as fibrin have been removed from cord blood.
According to a fourth aspect of the present invention, cord blood is obtained by a method of disrupting one or more blood cells or cells selected from the group consisting of freeze-thawing, freeze-drying, French press, X-press, and ultrasonic disruption. A hair growth agent characterized by containing a cord blood homogenate and / or a cord blood homogenate supernatant.

The fifth invention provides a placenta that can be obtained together with umbilical cord blood, a single cell component obtained by subjecting the placenta to extracellular matrix hydrolysis with trypsin and / or collagenase, placental homogenate, placental homogenate supernatant, placental blood, placental plasma, placental serum It is a hair growth agent characterized by containing one or two or more components selected from the group consisting of six.

The sixth invention is one selected from the group consisting of umbilical cord blood plasma, umbilical cord blood serum, umbilical cord blood homogenate supernatant, placental homogenate supernatant, placental blood plasma, placental blood serum, and / or two or more components Is a hair growth agent characterized by being dissolved in a nonionic aqueous solution, more preferably a nonionic surfactant aqueous solution, for iontophoresis.

The seventh invention is a single cell component obtained by subjecting placenta and / or umbilical cord blood to extracellular matrix hydrolyzate with trypsin and / or collagenase, cell surface antigen markers CD105 +, CD166 +, CD29 + from human umbilical cord blood using a Preparative Flow Cytometry device. Selected from the group consisting of umbilical cord blood-derived mesenchymal stem cell-rich fractions prepared using CD44 +, CD14-, CD34-, and CD45- as indicators, and / or two or more components for iontophoresis It is a hair growth agent characterized by being dissolved in a nonionic aqueous solution, more preferably a nonionic surfactant aqueous solution.

The eighth invention comprises one and / or two or more components selected from the group consisting of placenta and / or umbilical homogenate, placenta and / or umbilical homogenate supernatant, placental blood plasma, and placental blood serum. It is a hair growth agent characterized by being mixed with umbilical cord blood.

A ninth invention is characterized in that one and / or two or more components selected from the group consisting of autologous peripheral blood stem cells / stem cell homogenates / homogenate supernatants are mixed with umbilical cord blood. It is an agent.

A tenth invention is characterized in that umbilical cord blood and / or placental blood is included in liposomes for electroporation and / or dispersed in an isotonic solution or an aqueous solution within ± 15% of isotonic pressure. It is a hair growth agent.

In an eleventh aspect of the invention, umbilical cord blood and / or placental blood is an isotonic solution or an aqueous solution within ± 15% of isotonic pressure for microneedle injection, and / or mannitol, sucrose, gelatin, gelatin as a cell membrane stabilizer 50,000-400,000 ppm, more preferably 150,000, in an aqueous solution containing one and / or two or more ingredients selected from the group consisting of lysate, casein, casein lysate, provitamin C, provitamin E It is a hair growth agent characterized by being dispersed at 250,000 ppm.

In the twelfth and thirteenth inventions, umbilical cord blood that can be collected from about 100 ml from one newborn is mixed with umbilical cord blood for 3 to 50 people, more preferably 5 to 20 people, most preferably 7 to 10 people. Then, a cocktail umbilical cord blood is prepared, and the quality of each lot of umbilical cord blood is made uniform, and a hair growth agent and its production method.

In the fourteenth and fifteenth inventions, the connective tissue filled with two umbilical arteries contained in the umbilical cord and an extracellular matrix serving as a blood vessel gap such as one umbilical vein is used as a collagenous tissue and / or Alternatively, the mesenchymal stem cells present in Wharton's jelly have collagen and collagen fibers distributed vertically and horizontally in the extracellular matrix, and the background substrate is glucosaminoglycan, which is also an acidic mucopolysaccharide. A hair growth agent characterized by containing mesenchymal stem cell-rich umbilical cord blood prepared by concentrating a fraction that is protectively present in abundant mucus-like substances, and a method for producing the same.

The sixteenth and seventeenth aspects of the present invention are cells prepared from human umbilical cord blood that has been confirmed negative by performing a microbiological test including HIV-1, mycoplasma, hepatitis B / C virus, bacteria, yeast, and mold, using a Preparative Flow Cytometry device. For the human umbilical cord blood-derived mesenchymal stem cell rich fraction and / or the same mesenchymal stem cell rich fraction prepared using the surface antigen markers CD105 +, CD166 +, CD29 +, CD44 +, CD14-, CD34-, CD45- Umbilical cord-derived components called “collagenous tissue / collagen and acidic mucopolysaccharide glucosaminoglycan-rich mucus-like substance of umbilical cord extracellular matrix component called Wharton's jelly” and / or “extracted from placenta” It is a hair growth agent characterized in that it is composed of a mesenchymal stem cell-rich fraction with enhanced proliferation ability to which the "extracellular matrix component" has been added, and a method for producing the same.

Umbilical cord blood in this patent means fetal blood contained in the umbilical cord (umbilical cord: Saiai), a fetal tissue that connects the fetus and the mother. . It has been widely used since 1993 as a transplant medicine for patients with blood diseases such as leukemia. From the point of notation, “umbilical cord” is not a common Kanji character, so it is also described as “dial blood”.

The procedure for collecting umbilical cord blood is performed immediately after childbirth and after separating the newborn from the umbilical cord. After the birth of a newborn baby, after a certain period of time, the placenta may be “spontaneous” from the mother's body. In many cases, it is collected before the birth. The specific collection method varies slightly depending on the maternity hospital, but after birth, the umbilical cord is cut off at the part close to the newborn. The surface of the umbilical cord separated from the newborn is sterilized, and first, a collection needle connected to the collection bag with a tube is inserted into the umbilical cord blood vessel. The cord blood then flows out naturally by gravity without applying artificial pressure and is collected into a collection bag. In order to increase the umbilical cord blood collection rate and collect as much as possible, cut the umbilical cord as much as possible within the reasonable range, and then collect almost all of the umbilical cord blood while squeezing while sliding the tube. Devise as follows.

Collected umbilical cord blood is generally sent to the umbilical cord blood bank for distribution. In the cord blood bank, microorganisms and contamination are inspected, and after processing the cells, the cord blood is stored frozen in a liquid nitrogen tank and waits for the turn necessary for transplantation.

  In an umbilical cord blood bank that has been transported from an obstetric hospital where umbilical cord blood has been collected, cell treatment is performed, and then cryopreserved in liquid nitrogen. Umbilical cord blood first aggregates red blood cells by adding hydroxyethyl starch (HES). This is centrifuged or centrifuged using a special bag to remove excess red blood cells and plasma. A part of the test sample is taken out, and the remaining solution is added to a cryoprotectant in order to prevent damage caused by freezing, and then transferred to a freezing bag, and then cryopreserved. The reason why the cryoprotectant is necessary is that if cord blood is frozen as it is without any treatment, hematopoietic stem cells are killed by cell destruction or dehydration by ice crystals generated in the cells. When thawing and using umbilical cord blood that has been cryopreserved, it is transferred to the transplantation facility while being frozen in liquid nitrogen, and thawed quickly and gently with warm water at 37 ° C. immediately before transplantation. At that time, since the cryoprotective agent may have an adverse effect in the case of patients, particularly small children, it is preferable that the thawed umbilical cord blood is washed with a physiological isotonic solution and then used for transplantation. Umbilical cord blood transplantation is performed through the whole body by blood circulation through intravenous infusion, and hematopoietic stem cells that have reached the bone marrow are settled.

Cell components contained in the collected umbilical cord blood include not only hematopoietic stem cells but also red blood cells, white blood cells, platelets, plasma and the like. Red blood cells, platelets, and plasma are not necessary for transplantation, and at the time of transplantation, red blood cells that are variants of the ABO system are rather harmful to the side of patients who receive cord blood transplants. In addition, because the liquid nitrogen tank has limited space, it is necessary to exclude unnecessary components in order to use it effectively (Hiraku Hara, Umbilical cord blood transplantation, Shinsei Medical Publishing Co., Ltd., 2006, pp.30- 39, ISBN4-88002-654-9; Japan Cord Blood Bank NOW 42, 2012.4.23)).

  As for the amount of cord blood of a newborn, about 100 ml can be collected in a newborn baby weighing 3 kg, but this volume varies widely among individuals. The number of nucleated cells in umbilical cord blood is the highest in the order of 400 to 600 million, about 30%, and 6 to 800 million is also about 30%, and the third is 8 About 1% from the first billion is about 20%, and more than 10% is more than 1 billion (Norihiro Sato: “Standardization and safety of preserved umbilical cord blood for transplantation”: Special Issue 110, “Clinical Pathology”, Japanese Society of Clinical Pathology Edit, 1999, pp.28-33). More than 800 million nucleated cells are required for transplantation to larger adults. CD34 + cells in nucleated cells (young hematopoietic cells with low PDL levels that contain hematopoietic stem cells) are considered useful, but rarely contained in adult peripheral blood, but in the range of 0.2 to 0.5% in umbilical cord blood There are many examples included.

  Among the components of cord blood, hematopoietic stem cells have been attracting attention for many years, but mesenchymal stem cells were detected in cord blood as somatic stem cells different from hematopoietic stem cells. Traditionally, mesenchymal stem cells have been reported to be present in the bone marrow, but umbilical cord blood is also considered useful as a source. Mesenchymal stem cells can also differentiate into bone and cartilage, indicating the possibility of clinical application as regenerative medicine. Furthermore, the existence of progenitor cells that have multipotency into various tissues such as nerve cells, hepatocytes, and epithelial cells are suggested as non-artificial original human somatic cells similar to iPS cells, which are artificial products. Has been.

Umbilical cord blood is inferior to peripheral blood mobilized stem cells and bone marrow in terms of the number of cells and the speed of recovery and engraftment of hematopoiesis, but it is the best in the youthfulness of cells as a PDL level that is an indicator of growth potential ing. In addition, chronic GVHD, which is an index opposite to rejection, is useful because it has a small risk of complication associated with organ transplantation called graft versus host disease (Tetsuo Abe, “Hematopoietic Tumor”). Atlas, 4th revised edition, ISBN978-4-7849-4081-3, Nippon Medical Newspaper, 2009, pp.25-30; supervised by Shigetaka Asano, Yasuo Ikeda, Taku Uchiyama, "Miwa Hematology" Bunkodo, ISBN978 -4-7849-4081-3, 2006, pp.822-832, pp.833-839).

  One of the pillars of the present invention is mesenchymal stem cells concentrated from umbilical cord blood or fetus. Fetal-derived mesenchymal cells obtained by primary culture have a very high proliferative capacity, have a long life compared to bone marrow-derived mesenchymal cells, and have a large number of cell divisions before growth arrest, that is, a large growth potential. Mesenchymal cells are a generic term for bones, cartilage, adipose tissue, skeletal muscle, skin dermis, ligaments, tendons, and the like, which are known as connective tissue cells, but can also differentiate into hair roots and hair matrix cells. In ontogeny, it is a cell derived from the middle axis mesoderm. In addition to the midaxial mesoderm, there are developmentally visceral mesoderm-derived cells such as heart muscle, smooth muscle, and vascular endothelium.

  The surface of the umbilical cord is covered with amniotic monolayer cubic epithelial cells. The connective tissue that fills the extracellular matrix that becomes the space between blood vessels such as the two umbilical arteries contained in the umbilical cord and one umbilical vein is a connective tissue that can be said to be relatively primitive. Also called Wharton's jelly. The reticulocytic cells that develop processes are roughly the same as stromal cells and mesenchymal cells. They are rough and literally reticulated. Collagen and collagen fibers are distributed vertically and horizontally in the extracellular matrix, which is the background. The substrate is formed of a mucus-like substance rich in glucosaminoglycans, which are also acidic mucopolysaccharides.

  Similar tissue shape is not only present in such umbilical cords, but also in fetal connective tissue. Shortly after fertilization up to 6 months of fetal age, the dermis has a flexible shape like agar, and the dermis exhibits a state of a glue-like tissue. Mesenchymal cells contained in umbilical cord blood may have been collected in Wharton's jelly contained in the umbilical cord when collecting umbilical cord blood, and are truly contained in blood. It is skeptical whether or not it is. Although there is a possibility that it is derived from Wharton ’s jelly, it has been stated in the conventional research that mesenchymal cells are collected from umbilical cord blood at a certain rate. As one of the pillars of the present invention, a fraction obtained by densely collecting the mesenchymal stem cells is positioned as a hair growth agent.

  The definition of hair growth agent refers to a drug that increases hair that is effective for alopecia. Alopecia refers to a case where the hair density is reduced to 50% or less from the medical point of view, and it is also conspicuous even when viewed from others. There are many cases. From a physiological point of view, the shape of human hair varies depending on age and individual personality, and there is no guarantee that there is a prescribed hair state that this must be the case. The definition of alopecia refers to a condition in which the person himself was primarily expecting that the hair on the scalp that had grown in the past would continue to grow in the same way, and is commonly balding. ). In recent years, it is sometimes confused with laser hair removal for women, and for this reason it is sometimes called thin hair.

Hair growth means that the lost hair starts to grow again, and artificially promotes it. On the other hand, hair growth refers to growing hair that grows in the present situation or suppressing hair loss. The effects of hair growth agents can be broadly classified into five types. A hair growth agent that is suitable for and effective for male pattern baldness, commonly called M-type baldness, depends on the action of suppressing the action of male hormones. Regarding hair growth agents that work for scalp care, there are first types that improve scalp blood flow, followed by types that care for damaged scalp, and shampoos that have a hair-growth effect. There is also a type that prevents inactivation of hair matrix cells that play an indispensable role in the cycle in which hair grows. There are various forms, such as a hair growth agent that controls the action of male hormones, and a hair growth agent that improves the scalp environment. There is a type that suppresses the movement of 5α reductase, one of the male hormones that cause AGA. There is also a type that promotes hair growth by activating the hair papilla itself in the center of the hair root. There is a type that promotes the function of FGF (Fibroblast growth factor), a protein that controls hair growth, and firmly delivers the protein that transmits the hair growth command that has been dormant until now to the dermal papilla.
There is a type in which the growth period is extended and the scalp flexibly changes as the hair grows, and the hair grows easily.

There are various products on the market as hair growth agents, all of which are only drugs that exhibit life science and / or chemical effects, and cell preparations such as the present invention are unparalleled. Riup (registered trademark) based on the antihypertensive agent minoxidil, finasteride, an antiandrogen that inhibits 5-alpha reductase, an enzyme that converts testosterone into dihydrotestosterone, and pores filled with sebum (pores, sebaceous glands, Nano-impact (trademark) that breaks through the sweat pores and penetrates active ingredients, Medicinal Pure EX (trademark) with biopolyphosphate as one of the main components, and Kampo and Japanese-style plants such as Tamazaki ヅ rafu dialkanoide Medicinal scalp care GP lotion (trademark), Lilyge (trademark) centered on plant moisturizing ingredients mulberry white skin extract and paulownia leaf extract, Cytoline EX (trademark) containing CTP (cytopurine), and the like.

Human umbilical cord blood (called UB) confirmed negative by microbiological tests including HIV-1, mycoplasma, hepatitis B / C virus, bacteria, yeast, mold, Beckman
Hematopoietic stem cell rich fraction (referred to as HpS) prepared using Coulter's Preparative Flow Cytometry device with the cell surface antigen marker CD34 / CD133 as an indicator, cell surface antigen markers CD105 +, CD166 +, CD29 + Human umbilical cord blood-derived mesenchymal stem cell-rich fraction (McS), which is prepared using CD44 +, CD14-, CD34-, and CD45- as indicators, and the same mesenchymal stem cell-rich fraction are referred to as collagenous tissue / Wharton's jelly Reinforced umbilical cord extracellular matrix component and acidic mucopolysaccharide glucosaminoglycan rich mucus-like substance (referred to as Suppl-Ecm) enhanced mesenchymal stem cell rich fraction (McS-Ecm), Suppl- UB with Ecm added (referred to as UB-Ecm) is a circle with a radius of 40mm centered at a point of 140mm from the head to the face at the center of the frontal lobe of the frontal lobe of the scalp of alopecia subjects covering almost the entire scalp At 25mm intervals 10 mesotherapy syringes, 5 injections of UB, HpS, McS, McS-Ecm, and UB-Ecm for each of the five liquids. Treated 3 times a week for 5 weeks.

As a result, the number of hairs that grew per square centimeter, length (mm), and average hair diameter (μm) were measured with a fiber microscope. UB: 18 and 25; 4mm, 6 mm; 29 μm, 30 μm, HpS: 4, 2; 2 mm, 1 mm; 19 μm, 18 μm, McS: 78, 58; 7 mm, 5 mm; 33 μm, 46 μm,
McS-Ecm: 103, 85; 15mm,
16 mm; 59 μm, 61 μm, UB-Ecm: 26, 49; 5 mm, 7 mm; 34 μm, 41 μm. Suppl-Ecm was effective when added to McS, but was also effective when added to UB. McS was much more effective than UB, but HpS was not better than UB. Thus, McS and McS-Ecm of the present invention were excellent in the hair growth effect. It has been found that another UB of the present invention and UB-Ecm are effective for hair growth.

Claims (17)

A hair growth agent comprising umbilical cord blood. A hair growth agent comprising plasma from which blood cell components have been removed from umbilical cord blood. A hair growth agent comprising serum from which blood cell components and fiber proteins such as fibrin are removed from umbilical cord blood. Umbilical cord blood homogenate obtained by crushing one or more blood cells / cells selected from the group consisting of freeze-thawing, freeze-drying, French press, X-press, and ultrasonic disruption, and A hair growth agent comprising umbilical cord blood homogenate supernatant. A placenta that can be obtained with umbilical cord blood, a single cell component obtained by subjecting the placenta to extracellular matrix hydrolysis with trypsin and / or collagenase, placental homogenate, placental homogenate supernatant, placental blood, placental plasma, and placental serum. A hair growth agent comprising one or more selected components. One and / or two or more components selected from the group consisting of umbilical cord blood plasma, umbilical cord blood serum, umbilical cord blood homogenate supernatant, placental homogenate supernatant, placental blood plasma, and placental blood serum for iontophoresis A hair growth agent characterized by being dissolved in a nonionic aqueous solution, more preferably a nonionic surfactant aqueous solution. Single cell component of placenta and / or cord blood subjected to extracellular matrix hydrolyzate with trypsin and / or collagenase, cell surface antigen markers CD105 +, CD166 +, CD29 +, CD44 +, CD14−, from human umbilical cord blood with Preparative Flow Cytometry device One or more components selected from the group consisting of umbilical cord blood-derived mesenchymal stem cell-rich fractions prepared using CD34- and CD45- as indicators, and / or two or more components for nonionic aqueous solutions for iontophoresis, A hair growth agent that is preferably dissolved in a nonionic surfactant aqueous solution. Mixing one or two or more components selected from the group consisting of placenta and / or umbilical homogenate, placenta and / or umbilical homogenate supernatant, placental blood plasma, and placental blood serum with umbilical cord blood A hair growth agent characterized by. A hair growth agent characterized by mixing one or more components selected from the group consisting of autologous peripheral blood stem cells, stem cell homogenates, and homogenate supernatants into umbilical cord blood. A hair growth agent characterized in that umbilical cord blood and / or placental blood is included in liposomes for electroporation and / or dispersed in an isotonic solution or an aqueous solution within ± 15% of isotonic pressure. Umbilical cord blood and / or placental blood isotonic solution for microneedle injection or aqueous solution within ± 15% of isotonic pressure, and / or mannitol, sucrose, gelatin, gelatin lysate, casein, casein lysate as cell membrane stabilizer -Disperse in an aqueous solution containing one or two or more components selected from the group consisting of provitamin C and provitamin E, from 50,000 to 400,000 ppm, more preferably from 150,000 to 250,000 ppm. A hair growth agent characterized by. About umbilical cord blood that can be collected approximately 100 ml from one newborn, 3 to 50, more preferably 5 to 20, most preferably 7 to 10 umbilical cord blood is mixed to prepare cocktail umbilical cord blood, Hair growth agent characterized by uniformizing the quality of each lot of cord blood About umbilical cord blood that can be collected approximately 100 ml from one newborn, 3 to 50, more preferably 5 to 20, most preferably 7 to 10 umbilical cord blood is mixed to prepare cocktail umbilical cord blood, A method for producing a hair growth agent characterized by uniformizing the quality of each lot of cord blood. Two connective umbilical arteries contained in the umbilical cord and a connective tissue filling an extracellular matrix such as a umbilical vein such as a umbilical vein, in collagenous tissue and / or in Wharton's jelly The mesenchymal stem cells present in the extracellular matrix have collagen and collagen fibers distributed vertically and horizontally, and the background substrate is protected in a mucus-like substance rich in glucosaminoglycan, which is also an acidic mucopolysaccharide A hair growth agent comprising mesenchymal stem cell-rich umbilical cord blood prepared by concentrating an existing fraction. Two connective umbilical arteries contained in the umbilical cord and a connective tissue filling an extracellular matrix such as a umbilical vein such as a umbilical vein, in collagenous tissue and / or in Wharton's jelly The mesenchymal stem cells present in the extracellular matrix have collagen and collagen fibers distributed vertically and horizontally, and the background substrate is protected in a mucus-like substance rich in glucosaminoglycan, which is also an acidic mucopolysaccharide A method for producing a hair growth agent comprising mesenchymal stem cell-rich umbilical cord blood prepared by concentrating a fraction that is present in a natural manner. Preparative from human umbilical cord blood confirmed negative by microbiological tests including HIV-1, mycoplasma, hepatitis B / C virus, bacteria, yeast and mold
Human umbilical cord blood-derived mesenchymal stem cell rich fraction and / or the same mesenchymal stem cells prepared using the flow cytometer as a marker for cell surface antigen markers CD105 +, CD166 +, CD29 +, CD44 +, CD14-, CD34-, CD45- For the rich fraction, an umbilical-derived component called "collagenous and acidic mucopolysaccharide glucosaminoglycan-rich mucus-like substance of the umbilical cord extracellular matrix component called Wharton's jelly", and / or A hair growth agent comprising a mesenchymal stem cell rich fraction with enhanced proliferation ability added with "extracellular matrix component extracted from placenta" and a method for producing the same. Preparative from human umbilical cord blood confirmed negative by microbiological tests including HIV-1, mycoplasma, hepatitis B / C virus, bacteria, yeast and mold
Human umbilical cord blood-derived mesenchymal stem cell rich fraction and / or the same mesenchymal stem cells prepared using the flow cytometer as a marker for cell surface antigen markers CD105 +, CD166 +, CD29 +, CD44 +, CD14-, CD34-, CD45- For the rich fraction, an umbilical-derived component called "collagenous and acidic mucopolysaccharide glucosaminoglycan-rich mucus-like substance of the umbilical cord extracellular matrix component called Wharton's jelly", and / or A method for producing a hair growth agent comprising a mesenchymal stem cell rich fraction with enhanced proliferation ability to which "extracellular matrix component extracted from placenta" is added.